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HbA1c detection via high-sensitive boronate based surface plasmon resonance sensor
Sensors & Actuators: B. Chemical 306 (2020) 127561
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
HbA1c detection via high-sensitive boronate based surface plasmon resonance sensor
T
Merve Çalışır, Monireh Bakhshpour, Handan Yavuz, Adil Denizli* Hacettepe Universty, Faculty of Science, Department of Chemistry, Ankara, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Glycosylated hemoglobin Vinyl phenyl boronic acid Surface plasmon resonance Sensor Glucose Nanofilm
Glycosylated Hemoglobin, known as HbA1c, is the most commonly used molecule to track and diagnose the Type II diabetes and gives very convincing accurate results. Tracking the HbA1c is also an early diagnostic tool to indicate diabetes occurrence in high risk patience. Glycosylated hemoglobin is the result of glucose binding of the β-chain of hemoglobin to N-terminal valine and reflects the average glucose value over the past 2–3 months. HbA1c in the blood concentration of more than 141 mg/dL is often enough to diagnose diabetes. Boronic acid derivatives are often used in HbA1c determination due to the carbohydrate relationship based on the cis-diol interaction and determinations based on this association are mostly carried out by enzymatic sensors and HPLC. In addition to these methods, sensor studies have also started to be developed as an alternative. In this study, it is aimed to determine HbA1c by a surface plasmon resonance sensor (SPR) modified with a boronic acid derivative vinyl phenyl boronic acid. This study is shown that the pH value is an important parameter for binding and the signal received is increased as the concentration increases. Even at low concentrations like 10 μg/mL, signal can be received and it implies that more accurate measurement can be made at clinical concentration values which are higher. In artificial plasma studies, different sensograms are obtained for human serum albumin, immunoglobulin G, and hemoglobin molecules which all could bind to modified chip and the selectivity to the molecules are distinctively differentiated in comparison with HbA1c.
1. Introduction Diabetes is one of troubling and highly common disease that can be seen worldwide that causes abnormal glucose increase in the blood and tissues by disrupting the metabolism of fat, protein and carbohydrates in the presence of insufficient insulin or low levels of insulin in the body [1]. Two general types of the disease exist; Type I and Type II diabetes. In Type I diabetes, the beta cells in the pancreas cannot produce enough insulin to induce the passage of glucose from blood to cells. This version of the diabetes is congenital and treatment and treatment should be performed at the very early stages of human life. Type II diabetes is the most common type of diabetes worldwide and usually develops in the early 50 s with the reduction of physical movement and weight gain. In the course of the disease, hyperglycemia (high glucose level in the blood) cannot be prevented and pancreatic beta cells are exhausted [2]. HbA1c is a derivative of hemoglobin that occurs when exposed to high levels of blood sugar over a long period of time [3]. Once the hemoglobin protein has been glycosylated, erythrocytes remain the same in the blood throughout their lifetime until they are destroyed. Although HbA1c is not sufficient for direct diagnosis, it gives important
⁎
findings about the course of the disease [4]. In the many recent methods, measurement of HbA1c is considered to be the most reliable way of evaluating chronic hyperglycemia, and this measurement indicates the risk of developing Type II diabetes complications. The HbA1c diabetes limit is 48 mmol.mol−1 in IFCC unit or 6.5 % in the DCCT unit in the blood. HbA1c level is not affected by daily fluctuations in blood glucose concentration so it is accepted as a better diagnostic tool and biochemical marker of diabetes compared to other glucose clinical parameters because of this stability [5]. American Diabetes Association considers a HbA1c level below 7 % as normal [6]. Therefore, there is a need for development of tools for sensitive detection of diabetes and diabetes management. The levels of blood glucose can be measured with a handheld device provided by pharmacies. This test can be shown the levels of blood glucose in the diagnosis's time. But this test is not useful for managing diabetes [7,8]. There are different assay methods for measuring HbA1c, such as immunoassay, high-pressure liquid chromatography, spectrophotometry, ion-exchange and affinity chromatography, electrophoresis, boronate affinity chromatography, colorimetric methods, and mass spectroscopy [9–16]. Boron-polyol interactions are of great importance for human health,
Corresponding author. E-mail address: [email protected] (A. Denizli).
https://doi.org/10.1016/j.snb.2019.127561 Received 27 July 2019; Received in revised form 5 December 2019; Accepted 8 December 2019 Available online 09 December 2019 0925-4005/ © 2019 Published by Elsevier B.V.
Sensors & Actuators: B. Chemical 306 (2020) 127561
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2.4. Characterization of SPR chips
plant growth and the recognition of some bacteria. A boron-based probe prepared by targeting the sugars on the cell surface can recognize certain characteristic epitopes for the identification of diseases and can be of great benefit in early detection [17]. The specific interaction between boron and sugar molecules is the fundamental of this study by targeting HbA1c’s glucose bounded side with a boron chemical which is 4-vinylphenyl boronic acid (VPBA). For detecting this interaction, biosensors are recently developed as an alternative for traditional immunochemical assays and HPLC methods. Biosensors are defined as devices that carry out analyzes that involve a biological material such as tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, integrated with a physicochemical transducer that can be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical [18–21]. The electrochemical sensors for the detection of HbA1c reported in the literature generally have a LOD value between 0.024 % and 1.25 ug/mL, however all of them require a label or a redox indicator for measurement [22]. Surface plasmon resonance (SPR) sensor is optical sensing tools which provide real-time monitoring of interactions of a various template and chemical analytes SPR based sensors allow label-free detection of analytes with small sample volumes and they are advantegous because of their ability to handle complex samples and reusability. It allows highly specific measurement when a layer formed on the surface to selectively interact with the analyte [23–25]. The basis of this sensor dependent on measuring any shift in the resonance angle related to the mass of the bonded template at the SPR surfaces. In this article, gold surface of the SPR biosensor chip is modified with 4-vinyl phenyl boronic acid (VPBA) derivative to detect HbA1c molecules and results are discussed in terms of selectivity and concentration.
2.5. Kinetic studies
2. Experimental
2.6. Artificial plasma studies
2.1. Chemicals
Artificial plasma studies are based on comparison of the solutions used and the actual samples and it provide important information about the suitability of the system in practice. The solutions containing the HbA1c sample for the boronic acid based sensor chip are compared with the artificial plasma sample containing HbA1c.
Boronic acid based nanofilm coated and uncoated SPR chip surfaces are characterized with Fouirer transform infrared spectrophotometer attenuated total reflection spectrophotometer (FTIR-ATR), ellipsometer (Nanofilm EP3, Germany) and water contact angle measurements. FTIR-ATR characterization studies of boronic acid based nanofilm coated and uncoated chip surfaces are done by using the FTIR-ATR spectrophotometer (Thermo Fisher Scientific, Nicolet iS10, Waltham, MA, USA) in the wavenumber range of 400–4000 cm−1 at a resolution of 2 cm−1. An auto-nulling imaging ellipsometer is used for all thickness measurements of SPR chips. All measurements have been performed at a wavelength of 532 nm with an incidence angle of 50º.
After the preparation of boronic acid based nanofilm coated sensors, kinetic studies are started. The isoelectric point of HbA1c is taken from the literature as pI = 6.74. Based on this value, measurements are made at near pH ranges and optimum signal is obtained at pH 6. The samples are prepared in pH 6 phosphate buffer and solutions in a volume of 10 mL are prepared for the experiment. As the desorption solution, pH 7.4 phosphate buffer and 0.1 M NaCl solutions are used. Concentration scans are performed at a concentration range of 10−200 μg/mL. In the reusability studies, pH 6 buffer is passed for 3 min for equilibrium, then the sample at the same concentration is passed for 10 min and finally the desorption solution in the pH 7.4 buffer is passed for 3 min. This process is repeated 5 times and finally re-usability sensorgram is obtained.
Ethylene glycol dimethacrylate (EGDMA), 2-hydroxymethylmethacrylate (HEMA) polymerization, dipotassium hydrogen phosphate (K2HPO4) mono potassium phosphate (KH2PO4), allyl mercaptan and 4-vinyl phenyl boronic acid (VPBA) for surface modification are purchased from Sigma Aldrich (StLouis, USA). The initiator α, αazoisobutyronitrile (AIBN) used in the preparation of nanofilm is obtained from Fluka A.G. (Buchs, Switzerland) and the artificial human plasma are obtained from Tokra Medical (Ankara, Turkey). Surface plasmon resonance gold chips are obtained from GWC Technologies (Madison, Wisconsin, USA).
3. Results and discussion 3.1. Characterization of SPR chips FTIR spectrometer is chosen for surface characterization of VPBA coated chip between 4000−400 cm−1 bands and given in Fig. 1A. The presence of EGDMA in nanofilm structure is manifested by CeO vibrations at 1140 cm−1 peak. The OeH stretch is shown at 3467 cm−1. In addition, the peaks of 1450 and 2950 cm−1 are typical peaks for phenyl groups. Carbonyl stretch is seen at 1720 cm−1 and so is at 1375 cm−1. In addition, the BeC stretch is also given by confirming the presence of boronic-based polymer by giving a peak at 1140 cm−1 [27]. The thickness of the empty, only allyl mercaptan modified, VPBA uncoated nanofilm, and finally VPBA coated nanofilm SPR chips are measured by ellipsometry and shown in Fig. 1(B–E) and Table 1. The surface thickness of allyl mercaptane modified SPR chip is obtained 32 ± 1.2 nm by ellipsometry analysis. Also, the bare SPR chip surface are measured as 48 ± 2.1 nm and the nanofilm coated with VPBA SPR chip surface thickness are measured as 50 ± 4.1 nm. Based on these measurements, it is possible to say that nanofilms are applied to the surface homogeneously [28]. A liquid in contact with the solid surface forms a certain amount of an angle. This angle varies depending on the contacted solid and the contact fluid and good data for recognizing the surface. If the contact angle is greater than 90 degrees, it can be said that surface is hydrophobic. The contact angles of allyl mercaptan modified, VPBA uncoated nanofilm and VPBA coated nanofilm SPR chip surface s are taken and shown in Fig. 2 and Table 2.
2.2. Modification of SPR chips Before surface modification of the SPR chips, the gold surfaces are washed separately with 10 mL of pure ethyl alcohol, purified water and acidic piranha solution with sulfuric acid/hydrogen peroxide (3: 1 v/v) for 10 min. Then they are pressurized at 200 mmHg and allowed to dry under 37 ºC degree. Then, SPR gold chip surface is incubated for 12 h with 3 mM allyl mercaptan solution instilled. Then, it is purged with alcohol and vacuum dried at 220 mmHg and 25 ºC. 2.3. Preparation of SPR nanofilms A stock solution containing HEMA as monomer, EGDMA as crosslinker and 4-vinyl phenyl boronic acid (VPBA) is prepared. Subsequently, 0.1 mg AIBN is added to the solution as polymerization initiator and allowed to dissolve. It is kept in a nitrogenous medium, under 100 W and 365 nm UV light at for 20−25 min for polymerization (Scheme 1). After the expected polymerization is observed, the surface is washed with acetate buffer (25 mM and pH 5) and purified water [26]. 2
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Scheme 1. Schematic preparation of SPR nanofilm.
The allylation process on the blank chip surface and both the subsequent non-VBP-free nanofilm and the VBPA addition decrease the angle value in each step. In other words, all of the additions increase the hydrophilic properties of the surface.
LOQ = 10 S/m
where S is the standard deviation of the intercept and m is the slope of the regression line. LOD and LOQ values are found as 2.86 and 9.52 μg/ mL respectively. The detection of limit values of other studies is also given in Table 3.
3.2. Kinetic and isotherm analysis 3.2.1. Concentration effect on binding In these studies, HbA1c samples are prepared in pH 6.0 buffer solutions at 10, 50, 75, 100, 120, 150 and 200 μg/mL concentrations and their sensorgrams are taken and shown in Fig. 4 with the graph. Firstly, the pH 6.0 buffer solution is passed through the system for 5 min at room temperature, then one of the HbaA1c solutions at the specified concentrations is passed for 8 min, then the pH 7.4 desorption solution is removed with 2 min. As the HbA1c concentration increases, the ΔR value is clearly increased, as is seen in 7 different samples made between 10 μg/mL-200 μg/mL concentrations at room temperature (Fig. 3). The equation of the linear graph taken from the concentration range of 10−200 μg/mL is calculated as y = 0.0548x-1.6212. The linearity of this equation is obtained as R2 = 0.9471. Since R2 is proportional to binding, binding is determined as 98.67 % linearity. All the detection experiments are done in replicates. For the repetitive experiments and the statistical experimental results, standard statistical methods are used to determine the mean values and relative standard deviations (RSD). Confidence intervals of 96 % are calculated for each set of samples in order to determine the margin of error. The limit of detection (LOD) and limit of quantification (LOQ) values were confirmed according to Eqs. 1, and 2. LOD = 3 S/m
(2)
3.2.2. Equilibrium Analysis and Association kinetic analysis The equilibrium analyzes are examined by Scatchard isotherm. Scatchard analysis is a graph that can determine the amount of concentration between the binding ligand and the non-binding ligand. Scatchard graph calculates the concentration of the substance, which is bound and not bound in the surface plasmon resonance sensors, on the basis of diffraction change. In the Scatchard equation, dΔR/dt = kaC (ΔRmax- ΔR)kdΔR
(3)
In the equation, kd and ka are the dissociation- and association rate. The ratio of these to each other is ka / kd, which gives the KA value which is the binding constant of the reaction. Since the chip surface will remain stable in balance, no change in the angle of the light can be expected to be observed. Therefore, since the change in diffraction is zero, d ΔR/dt is equal to zero. When this equality is simplified, the following equality is obtained for calculations. ΔReq/C = KAΔRmax– KAΔReq
(4)
In the Scathard graph that is obtained from concentration measurement, x-axis intercept gives the association constant and y-axis intercept gives the dissociation constant. Assuming that a binding occurs from a point where ΔR is the maximum, the KA and KD values can
(1) 3
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Fig. 1. A. FTIR spectrum of VPBA coated nanofilm SPR chip. Ellipsometry images of bare SPR chip surface (B), allyl mercaptan modified SPR chip surface (C), VPBA uncoated nanofilm SPR chip surface (D), and the VPBA-coated nanofilm SPR chip surface (E).
dΔR/dt = kaCΔRmax-(kaC + kd)ΔR
Table 1 Ellipsometry values of SPR chip surfaces. Surface
Thickness (nm)
Allyl mercaptan modified SPR chip surface VPBA uncoated nanofilm SPR chip surface VPBA coated nanofilm SPR chip surface
32 ± 1.2 48 ± 2.1 50 ± 4.1
(5)
The slope of the obtained correct graph gives the relationship between the binding speed and analyte concentration and can be used to calculate the amount. By using Rmax value in equality, kd and ka values can be calculated. Since the amount of sample to be used to fill the sensor surface is too much, it will be possible to calculate the forward and reverse rate constants from the ΔR and dΔR/dt graphs of the sensorgrams to be taken at different sample concentrations.
be obtained by using Eq. (4). When the associating kinetics is applied for equilibrium, an analysis can be performed by reorganizing the Eq. (3).
S = kaC + kd
(6)
The slope of the line given in Eq. 6 is ka and intercept gives kd. At a 4
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Fig. 2. A. Allyl mercaptan modified, B. VPBA uncoated nanofilm and C. VPBA coated nanofilm SPR chip surfaces contact angle images.
specified starting point, it would be difficult to calculate the kd value because the binding constant would be much higher than the dissociation constant. The natural logarithm of ΔR0 and ΔRt diffraction rates at the specified time t0 and t gives more definite result in calculating the kd value.
Table 2 Water contact angle values of SPR chip surfaces. Surface
Water Contact Angle
Empty Chip Allyl mercaptan modified chip surface VPBA uncoated nanofilm chip surface VPBA nanofilm chip surface
81.4 81.1 68.1 55.2
± ± ± ±
0
0.18 0.23 0.30 0.12
ln(ΔRt0/ΔRt) = kd (t – t0)
(7)
All binding kinetics parameters are calculated from these equations and given in the Table 4. When the results of the binding kinetics analysis are examined, the higher the KA value and the lower the KD values are expected, because it is the evidence that the analyses shows high affinity to the receptor. In this graphs that are obtained from experiment, the KA value is 43 μg/mL and the KD value is 0.0233 mL/μg. The ratio of the two ratios to each other is seen to be high and it is one of the proofs that HbA1c is successfully connected to the modified sensor.
3.2.3. Isotherm analysis The Freundlich model is an isotherm model for adsorption on heterogeneous surfaces. By adapting the standard parameters for Surface plasmon resonance, the following formula is obtained. ΔR=ΔRmax[C]1/n
(8)
Langmuir isotherm is obtained by assuming that the molecules on the surface are adsorbed uniformly. The formulation of Langmuir isotherm equations adapted to the surface plasmon resonance sensor ΔR value is in below. ΔR={ΔRmax[C]/(KD+[C])}
(9)
A combination of Langmuir-Freundlich isotherm model is also used by the formulation given below. ΔR={ΔRmax[C]1/n/KD+[C]1/n}
(10)
Freundlich, Langmuir and Freundlich-Langmuir isotherms are taken in order to examine the interaction between HbA1c and boronic acid based sensor system and given in Table 5. A successful binding is observed in all of the isotherm models that obtained. However, especially the Langmuir model with ΔRmax equals 12.787 is found to be the most appropriate model to identify the interaction between VPBA nanofilm SPR sensor and HbA1c molecule.
Fig. 3. Sensorgrams and graph of the interaction between HbA1c solutions and SPR sensors at different concentrations, n: 3.
5
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Fig. 4. A.B.C The sensograms of comparison of HbA1c binding signal by IgG, hemoglobin and human serum albumin at a concentration of 120, 150, 200 μg/mL, and D. taken by adding HbA1c at a concentration of 30, 50, 120 μg/mL in artificial plasma, n: 3. Table 3 The limit of detection values of HbA1c determination sensors by using boronic acid derivatives. Boronic Acid Derivative
Modification agent or electrode
Detection Method
Limit of Detection
Ref.
T3BA APBA APBA APBA APBA VPBA
Au/IgG-FITC glutaraldehyde-SAM Cys glutaraldehyde-ESMs pTTBA-Au NPs PQQ-ERGO/GCE VPBA-EGDMA-HEMA
Impedance Impedance Impedance Amperometric Voltammetry SPR
1 % 0.024 %0.19–0.21 % 0.052 1.25 2.86
[29] [30] [31] [32] [33] In this study
Table 4 Parameters of equilibrium and association kinetic analysis. Scatchard Equilibrium Analysis
Association Kinetic Analysis
ΔRmax KA (μg/mL) KD (mL/μg) R2
ka, (μg/mL)−1s−1 kd, s−1 KA (μg/mL) KD (mL/μg) R2
9.66 0.007 142.857 0.8633
Table 5 Calculated Freundlich, Parameters. Freundlich ΔRmax 1/n R2
Langmuir
and
Langmuir-Freundlich
ΔRmax KD,(mL/μg) KA,(μg/mL) R2
VPBA coated SPR Sensor 0.0005 0.0215 43.000 0.0233 0.9471
HbA1C IgG HSA Hb
VPBA uncoated SPR Sensor
ΔR
k
ΔR
k
k'
5.2 0.71 0.3 0.15
7.32 17.33 34.66
0.15 0.11 0.1 0.1
1.36 1.5 1.5
5.38 11.55 23.11
Isotherm
3.3. Selectivity studies
Langmuir 2.977 1.27 0.8573
Table 6 Comparative selectivity coefficients for IgG, HSA, hemoglobin and HbA1c with VPBA-coated and uncoated SPR sensors.
Langmuir -Freundlich 12.787 601.023 0.00166 0.9288
ΔRmax 1/n KD,(mL/μg) KA,(μg/mL) R2
Measurements are made with artificial plasma to determine the selectivity of the boronic acid-based surface plasmon resonance sensor. Measurements are made to see whether the high affinity based on the cis-diol interaction of HbA1c to boronic acid shows equally high affinity for the IgG, HSA and Hb in plasma. The sensogram of all the different samples at a concentration of 120, 150, 200 μg/mL and the sensograms obtained by adding HbA1c at a concentration of 30, 50, 120 μg/mL to the artificial plasma are obtained and shown in Fig. 4. The comparison experiment of HbA1c binding signal with IgG, Hb, and HSA are
0.1105 9.0506 18.1811 0.05500 0.9244
6
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Fig. 5. Reusability sensogram of VPBA coated SPR sensor in HbA1c measurement.
conducted at the different concentration. According to the results, there are neglectable interaction between boronic acid and compare molecules. Selectivity coefficient (k) and relative selectivity coefficient (k´) of the prepared sensor are determined by subtracting from the 6th and 7th equations and shown in Table 6. k=ΔRHbA1c/ΔRcompetitor
(11)
k´=kVPBA/kempty
(12)
For HbA1c, the VPBA spiked sensor showed 7.32 times more selectivity than IgG and also showed 17.33 and 34.66 more selectivity according to HSA and Hb respectively. Additionally, when the ΔR comparison with the unmodified chip is examined, a serious difference of 5.2/0.15 is observed, which proves that nanofilm is successfully established high affinity with HbA1c. 3.4. Reusability studies In the reusability study, HbA1c solution in pH 6.0 buffer at a concentration of 75 μg/mL is given to the system at room temperature. After passing through the buffer system, the sample is given for about 5 min and after 3 min the desorption solution is passed, the same procedure is repeated 5 times and shown in Fig. 5. As shown in the figure, almost the same ΔR value is obtained for each repeated sample. 4. Conclusion Studies have been conducted to determine the glycosylated hemoglobin (HbA1c), which is a vital measure for diabetes detection and early diagnosis, with the SPR sensor. Because the cis-diol interactions between glucose and boronic acid are predicted to be unique and potent, vinyl phenyl boronic acid (VPBA) bound coated nanofilm is synthesized and the relationship between glycosylated hemoglobin is observed by experiments. All characterization studies and subsequent kinetic studies, equilibrium analysis and isotherms proves that the desired binding is occurred. The detection limit for the study is calculated as 2.86 μg/mL. Although this value is higher than the calculated limit values in other studies, it can be lowered by the concentration and device changes in the calibration stage. Finally, the proposed approach can perform measurements far below the aforementioned clinical values proving the validity of the study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 7
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purification and isolation of biomolecules.
Merve Çalışır received master degree from Hacettepe University, Turkey in 2019. She is working on optical sensor system, protein detection, chromatographic separation of biomolecules and biosensors.
Adil Denizli (PhD) is a professor at Hacettepe University, Ankara, Turkey. He received PhD degree from the same university in 1992. He is a professor at the chemistry department, Hacettepe University since 2000 and now he is also the head of the Biochemistry Division. His main research fields are molecular imprinting technologies, hemoperfusion, removal of toxic materials from blood, purification of enzymes and proteins by chromatographic methods, biosensors based (SPR, QCM) on synthetic receptor technology, production of polymers havedifferent surface and bulk properties, shape and geometries, application of these polymers in medicine and biology.
Handan Yavuz (PhD) is a professor at Hacettepe University, Ankara, Turkey. She received PhD degree from the same university in 2003. She is a professor at Chemistry department, Hacettepe University. Her main research fields are preparation of polymeric biospecific sorbents and their use for the purification of enzymes and proteins, production of polymeric biomaterials, characterization of bulk and surface properties and modification of their surfaces by various methods.
Monireh Bakhshpour (PhD) received the master degree and PhD degree in 2011 and 2015 at Biochemistry Division of Chemistry Department, Hacettepe University respectively. She is working on designed and developed Surface Plasmon Resonance (SPR), and Quartz Crystal Microbalance (QCM) based chips for detection of pathogenic microorganisms, affinity chromatography, synthesis and characterization of polymeric matrix,
8
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
HbA1c detection via high-sensitive boronate based surface plasmon resonance sensor
T
Merve Çalışır, Monireh Bakhshpour, Handan Yavuz, Adil Denizli* Hacettepe Universty, Faculty of Science, Department of Chemistry, Ankara, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Glycosylated hemoglobin Vinyl phenyl boronic acid Surface plasmon resonance Sensor Glucose Nanofilm
Glycosylated Hemoglobin, known as HbA1c, is the most commonly used molecule to track and diagnose the Type II diabetes and gives very convincing accurate results. Tracking the HbA1c is also an early diagnostic tool to indicate diabetes occurrence in high risk patience. Glycosylated hemoglobin is the result of glucose binding of the β-chain of hemoglobin to N-terminal valine and reflects the average glucose value over the past 2–3 months. HbA1c in the blood concentration of more than 141 mg/dL is often enough to diagnose diabetes. Boronic acid derivatives are often used in HbA1c determination due to the carbohydrate relationship based on the cis-diol interaction and determinations based on this association are mostly carried out by enzymatic sensors and HPLC. In addition to these methods, sensor studies have also started to be developed as an alternative. In this study, it is aimed to determine HbA1c by a surface plasmon resonance sensor (SPR) modified with a boronic acid derivative vinyl phenyl boronic acid. This study is shown that the pH value is an important parameter for binding and the signal received is increased as the concentration increases. Even at low concentrations like 10 μg/mL, signal can be received and it implies that more accurate measurement can be made at clinical concentration values which are higher. In artificial plasma studies, different sensograms are obtained for human serum albumin, immunoglobulin G, and hemoglobin molecules which all could bind to modified chip and the selectivity to the molecules are distinctively differentiated in comparison with HbA1c.
1. Introduction Diabetes is one of troubling and highly common disease that can be seen worldwide that causes abnormal glucose increase in the blood and tissues by disrupting the metabolism of fat, protein and carbohydrates in the presence of insufficient insulin or low levels of insulin in the body [1]. Two general types of the disease exist; Type I and Type II diabetes. In Type I diabetes, the beta cells in the pancreas cannot produce enough insulin to induce the passage of glucose from blood to cells. This version of the diabetes is congenital and treatment and treatment should be performed at the very early stages of human life. Type II diabetes is the most common type of diabetes worldwide and usually develops in the early 50 s with the reduction of physical movement and weight gain. In the course of the disease, hyperglycemia (high glucose level in the blood) cannot be prevented and pancreatic beta cells are exhausted [2]. HbA1c is a derivative of hemoglobin that occurs when exposed to high levels of blood sugar over a long period of time [3]. Once the hemoglobin protein has been glycosylated, erythrocytes remain the same in the blood throughout their lifetime until they are destroyed. Although HbA1c is not sufficient for direct diagnosis, it gives important
⁎
findings about the course of the disease [4]. In the many recent methods, measurement of HbA1c is considered to be the most reliable way of evaluating chronic hyperglycemia, and this measurement indicates the risk of developing Type II diabetes complications. The HbA1c diabetes limit is 48 mmol.mol−1 in IFCC unit or 6.5 % in the DCCT unit in the blood. HbA1c level is not affected by daily fluctuations in blood glucose concentration so it is accepted as a better diagnostic tool and biochemical marker of diabetes compared to other glucose clinical parameters because of this stability [5]. American Diabetes Association considers a HbA1c level below 7 % as normal [6]. Therefore, there is a need for development of tools for sensitive detection of diabetes and diabetes management. The levels of blood glucose can be measured with a handheld device provided by pharmacies. This test can be shown the levels of blood glucose in the diagnosis's time. But this test is not useful for managing diabetes [7,8]. There are different assay methods for measuring HbA1c, such as immunoassay, high-pressure liquid chromatography, spectrophotometry, ion-exchange and affinity chromatography, electrophoresis, boronate affinity chromatography, colorimetric methods, and mass spectroscopy [9–16]. Boron-polyol interactions are of great importance for human health,
Corresponding author. E-mail address: [email protected] (A. Denizli).
https://doi.org/10.1016/j.snb.2019.127561 Received 27 July 2019; Received in revised form 5 December 2019; Accepted 8 December 2019 Available online 09 December 2019 0925-4005/ © 2019 Published by Elsevier B.V.
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2.4. Characterization of SPR chips
plant growth and the recognition of some bacteria. A boron-based probe prepared by targeting the sugars on the cell surface can recognize certain characteristic epitopes for the identification of diseases and can be of great benefit in early detection [17]. The specific interaction between boron and sugar molecules is the fundamental of this study by targeting HbA1c’s glucose bounded side with a boron chemical which is 4-vinylphenyl boronic acid (VPBA). For detecting this interaction, biosensors are recently developed as an alternative for traditional immunochemical assays and HPLC methods. Biosensors are defined as devices that carry out analyzes that involve a biological material such as tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, integrated with a physicochemical transducer that can be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical [18–21]. The electrochemical sensors for the detection of HbA1c reported in the literature generally have a LOD value between 0.024 % and 1.25 ug/mL, however all of them require a label or a redox indicator for measurement [22]. Surface plasmon resonance (SPR) sensor is optical sensing tools which provide real-time monitoring of interactions of a various template and chemical analytes SPR based sensors allow label-free detection of analytes with small sample volumes and they are advantegous because of their ability to handle complex samples and reusability. It allows highly specific measurement when a layer formed on the surface to selectively interact with the analyte [23–25]. The basis of this sensor dependent on measuring any shift in the resonance angle related to the mass of the bonded template at the SPR surfaces. In this article, gold surface of the SPR biosensor chip is modified with 4-vinyl phenyl boronic acid (VPBA) derivative to detect HbA1c molecules and results are discussed in terms of selectivity and concentration.
2.5. Kinetic studies
2. Experimental
2.6. Artificial plasma studies
2.1. Chemicals
Artificial plasma studies are based on comparison of the solutions used and the actual samples and it provide important information about the suitability of the system in practice. The solutions containing the HbA1c sample for the boronic acid based sensor chip are compared with the artificial plasma sample containing HbA1c.
Boronic acid based nanofilm coated and uncoated SPR chip surfaces are characterized with Fouirer transform infrared spectrophotometer attenuated total reflection spectrophotometer (FTIR-ATR), ellipsometer (Nanofilm EP3, Germany) and water contact angle measurements. FTIR-ATR characterization studies of boronic acid based nanofilm coated and uncoated chip surfaces are done by using the FTIR-ATR spectrophotometer (Thermo Fisher Scientific, Nicolet iS10, Waltham, MA, USA) in the wavenumber range of 400–4000 cm−1 at a resolution of 2 cm−1. An auto-nulling imaging ellipsometer is used for all thickness measurements of SPR chips. All measurements have been performed at a wavelength of 532 nm with an incidence angle of 50º.
After the preparation of boronic acid based nanofilm coated sensors, kinetic studies are started. The isoelectric point of HbA1c is taken from the literature as pI = 6.74. Based on this value, measurements are made at near pH ranges and optimum signal is obtained at pH 6. The samples are prepared in pH 6 phosphate buffer and solutions in a volume of 10 mL are prepared for the experiment. As the desorption solution, pH 7.4 phosphate buffer and 0.1 M NaCl solutions are used. Concentration scans are performed at a concentration range of 10−200 μg/mL. In the reusability studies, pH 6 buffer is passed for 3 min for equilibrium, then the sample at the same concentration is passed for 10 min and finally the desorption solution in the pH 7.4 buffer is passed for 3 min. This process is repeated 5 times and finally re-usability sensorgram is obtained.
Ethylene glycol dimethacrylate (EGDMA), 2-hydroxymethylmethacrylate (HEMA) polymerization, dipotassium hydrogen phosphate (K2HPO4) mono potassium phosphate (KH2PO4), allyl mercaptan and 4-vinyl phenyl boronic acid (VPBA) for surface modification are purchased from Sigma Aldrich (StLouis, USA). The initiator α, αazoisobutyronitrile (AIBN) used in the preparation of nanofilm is obtained from Fluka A.G. (Buchs, Switzerland) and the artificial human plasma are obtained from Tokra Medical (Ankara, Turkey). Surface plasmon resonance gold chips are obtained from GWC Technologies (Madison, Wisconsin, USA).
3. Results and discussion 3.1. Characterization of SPR chips FTIR spectrometer is chosen for surface characterization of VPBA coated chip between 4000−400 cm−1 bands and given in Fig. 1A. The presence of EGDMA in nanofilm structure is manifested by CeO vibrations at 1140 cm−1 peak. The OeH stretch is shown at 3467 cm−1. In addition, the peaks of 1450 and 2950 cm−1 are typical peaks for phenyl groups. Carbonyl stretch is seen at 1720 cm−1 and so is at 1375 cm−1. In addition, the BeC stretch is also given by confirming the presence of boronic-based polymer by giving a peak at 1140 cm−1 [27]. The thickness of the empty, only allyl mercaptan modified, VPBA uncoated nanofilm, and finally VPBA coated nanofilm SPR chips are measured by ellipsometry and shown in Fig. 1(B–E) and Table 1. The surface thickness of allyl mercaptane modified SPR chip is obtained 32 ± 1.2 nm by ellipsometry analysis. Also, the bare SPR chip surface are measured as 48 ± 2.1 nm and the nanofilm coated with VPBA SPR chip surface thickness are measured as 50 ± 4.1 nm. Based on these measurements, it is possible to say that nanofilms are applied to the surface homogeneously [28]. A liquid in contact with the solid surface forms a certain amount of an angle. This angle varies depending on the contacted solid and the contact fluid and good data for recognizing the surface. If the contact angle is greater than 90 degrees, it can be said that surface is hydrophobic. The contact angles of allyl mercaptan modified, VPBA uncoated nanofilm and VPBA coated nanofilm SPR chip surface s are taken and shown in Fig. 2 and Table 2.
2.2. Modification of SPR chips Before surface modification of the SPR chips, the gold surfaces are washed separately with 10 mL of pure ethyl alcohol, purified water and acidic piranha solution with sulfuric acid/hydrogen peroxide (3: 1 v/v) for 10 min. Then they are pressurized at 200 mmHg and allowed to dry under 37 ºC degree. Then, SPR gold chip surface is incubated for 12 h with 3 mM allyl mercaptan solution instilled. Then, it is purged with alcohol and vacuum dried at 220 mmHg and 25 ºC. 2.3. Preparation of SPR nanofilms A stock solution containing HEMA as monomer, EGDMA as crosslinker and 4-vinyl phenyl boronic acid (VPBA) is prepared. Subsequently, 0.1 mg AIBN is added to the solution as polymerization initiator and allowed to dissolve. It is kept in a nitrogenous medium, under 100 W and 365 nm UV light at for 20−25 min for polymerization (Scheme 1). After the expected polymerization is observed, the surface is washed with acetate buffer (25 mM and pH 5) and purified water [26]. 2
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Scheme 1. Schematic preparation of SPR nanofilm.
The allylation process on the blank chip surface and both the subsequent non-VBP-free nanofilm and the VBPA addition decrease the angle value in each step. In other words, all of the additions increase the hydrophilic properties of the surface.
LOQ = 10 S/m
where S is the standard deviation of the intercept and m is the slope of the regression line. LOD and LOQ values are found as 2.86 and 9.52 μg/ mL respectively. The detection of limit values of other studies is also given in Table 3.
3.2. Kinetic and isotherm analysis 3.2.1. Concentration effect on binding In these studies, HbA1c samples are prepared in pH 6.0 buffer solutions at 10, 50, 75, 100, 120, 150 and 200 μg/mL concentrations and their sensorgrams are taken and shown in Fig. 4 with the graph. Firstly, the pH 6.0 buffer solution is passed through the system for 5 min at room temperature, then one of the HbaA1c solutions at the specified concentrations is passed for 8 min, then the pH 7.4 desorption solution is removed with 2 min. As the HbA1c concentration increases, the ΔR value is clearly increased, as is seen in 7 different samples made between 10 μg/mL-200 μg/mL concentrations at room temperature (Fig. 3). The equation of the linear graph taken from the concentration range of 10−200 μg/mL is calculated as y = 0.0548x-1.6212. The linearity of this equation is obtained as R2 = 0.9471. Since R2 is proportional to binding, binding is determined as 98.67 % linearity. All the detection experiments are done in replicates. For the repetitive experiments and the statistical experimental results, standard statistical methods are used to determine the mean values and relative standard deviations (RSD). Confidence intervals of 96 % are calculated for each set of samples in order to determine the margin of error. The limit of detection (LOD) and limit of quantification (LOQ) values were confirmed according to Eqs. 1, and 2. LOD = 3 S/m
(2)
3.2.2. Equilibrium Analysis and Association kinetic analysis The equilibrium analyzes are examined by Scatchard isotherm. Scatchard analysis is a graph that can determine the amount of concentration between the binding ligand and the non-binding ligand. Scatchard graph calculates the concentration of the substance, which is bound and not bound in the surface plasmon resonance sensors, on the basis of diffraction change. In the Scatchard equation, dΔR/dt = kaC (ΔRmax- ΔR)kdΔR
(3)
In the equation, kd and ka are the dissociation- and association rate. The ratio of these to each other is ka / kd, which gives the KA value which is the binding constant of the reaction. Since the chip surface will remain stable in balance, no change in the angle of the light can be expected to be observed. Therefore, since the change in diffraction is zero, d ΔR/dt is equal to zero. When this equality is simplified, the following equality is obtained for calculations. ΔReq/C = KAΔRmax– KAΔReq
(4)
In the Scathard graph that is obtained from concentration measurement, x-axis intercept gives the association constant and y-axis intercept gives the dissociation constant. Assuming that a binding occurs from a point where ΔR is the maximum, the KA and KD values can
(1) 3
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Fig. 1. A. FTIR spectrum of VPBA coated nanofilm SPR chip. Ellipsometry images of bare SPR chip surface (B), allyl mercaptan modified SPR chip surface (C), VPBA uncoated nanofilm SPR chip surface (D), and the VPBA-coated nanofilm SPR chip surface (E).
dΔR/dt = kaCΔRmax-(kaC + kd)ΔR
Table 1 Ellipsometry values of SPR chip surfaces. Surface
Thickness (nm)
Allyl mercaptan modified SPR chip surface VPBA uncoated nanofilm SPR chip surface VPBA coated nanofilm SPR chip surface
32 ± 1.2 48 ± 2.1 50 ± 4.1
(5)
The slope of the obtained correct graph gives the relationship between the binding speed and analyte concentration and can be used to calculate the amount. By using Rmax value in equality, kd and ka values can be calculated. Since the amount of sample to be used to fill the sensor surface is too much, it will be possible to calculate the forward and reverse rate constants from the ΔR and dΔR/dt graphs of the sensorgrams to be taken at different sample concentrations.
be obtained by using Eq. (4). When the associating kinetics is applied for equilibrium, an analysis can be performed by reorganizing the Eq. (3).
S = kaC + kd
(6)
The slope of the line given in Eq. 6 is ka and intercept gives kd. At a 4
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Fig. 2. A. Allyl mercaptan modified, B. VPBA uncoated nanofilm and C. VPBA coated nanofilm SPR chip surfaces contact angle images.
specified starting point, it would be difficult to calculate the kd value because the binding constant would be much higher than the dissociation constant. The natural logarithm of ΔR0 and ΔRt diffraction rates at the specified time t0 and t gives more definite result in calculating the kd value.
Table 2 Water contact angle values of SPR chip surfaces. Surface
Water Contact Angle
Empty Chip Allyl mercaptan modified chip surface VPBA uncoated nanofilm chip surface VPBA nanofilm chip surface
81.4 81.1 68.1 55.2
± ± ± ±
0
0.18 0.23 0.30 0.12
ln(ΔRt0/ΔRt) = kd (t – t0)
(7)
All binding kinetics parameters are calculated from these equations and given in the Table 4. When the results of the binding kinetics analysis are examined, the higher the KA value and the lower the KD values are expected, because it is the evidence that the analyses shows high affinity to the receptor. In this graphs that are obtained from experiment, the KA value is 43 μg/mL and the KD value is 0.0233 mL/μg. The ratio of the two ratios to each other is seen to be high and it is one of the proofs that HbA1c is successfully connected to the modified sensor.
3.2.3. Isotherm analysis The Freundlich model is an isotherm model for adsorption on heterogeneous surfaces. By adapting the standard parameters for Surface plasmon resonance, the following formula is obtained. ΔR=ΔRmax[C]1/n
(8)
Langmuir isotherm is obtained by assuming that the molecules on the surface are adsorbed uniformly. The formulation of Langmuir isotherm equations adapted to the surface plasmon resonance sensor ΔR value is in below. ΔR={ΔRmax[C]/(KD+[C])}
(9)
A combination of Langmuir-Freundlich isotherm model is also used by the formulation given below. ΔR={ΔRmax[C]1/n/KD+[C]1/n}
(10)
Freundlich, Langmuir and Freundlich-Langmuir isotherms are taken in order to examine the interaction between HbA1c and boronic acid based sensor system and given in Table 5. A successful binding is observed in all of the isotherm models that obtained. However, especially the Langmuir model with ΔRmax equals 12.787 is found to be the most appropriate model to identify the interaction between VPBA nanofilm SPR sensor and HbA1c molecule.
Fig. 3. Sensorgrams and graph of the interaction between HbA1c solutions and SPR sensors at different concentrations, n: 3.
5
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Fig. 4. A.B.C The sensograms of comparison of HbA1c binding signal by IgG, hemoglobin and human serum albumin at a concentration of 120, 150, 200 μg/mL, and D. taken by adding HbA1c at a concentration of 30, 50, 120 μg/mL in artificial plasma, n: 3. Table 3 The limit of detection values of HbA1c determination sensors by using boronic acid derivatives. Boronic Acid Derivative
Modification agent or electrode
Detection Method
Limit of Detection
Ref.
T3BA APBA APBA APBA APBA VPBA
Au/IgG-FITC glutaraldehyde-SAM Cys glutaraldehyde-ESMs pTTBA-Au NPs PQQ-ERGO/GCE VPBA-EGDMA-HEMA
Impedance Impedance Impedance Amperometric Voltammetry SPR
1 % 0.024 %0.19–0.21 % 0.052 1.25 2.86
[29] [30] [31] [32] [33] In this study
Table 4 Parameters of equilibrium and association kinetic analysis. Scatchard Equilibrium Analysis
Association Kinetic Analysis
ΔRmax KA (μg/mL) KD (mL/μg) R2
ka, (μg/mL)−1s−1 kd, s−1 KA (μg/mL) KD (mL/μg) R2
9.66 0.007 142.857 0.8633
Table 5 Calculated Freundlich, Parameters. Freundlich ΔRmax 1/n R2
Langmuir
and
Langmuir-Freundlich
ΔRmax KD,(mL/μg) KA,(μg/mL) R2
VPBA coated SPR Sensor 0.0005 0.0215 43.000 0.0233 0.9471
HbA1C IgG HSA Hb
VPBA uncoated SPR Sensor
ΔR
k
ΔR
k
k'
5.2 0.71 0.3 0.15
7.32 17.33 34.66
0.15 0.11 0.1 0.1
1.36 1.5 1.5
5.38 11.55 23.11
Isotherm
3.3. Selectivity studies
Langmuir 2.977 1.27 0.8573
Table 6 Comparative selectivity coefficients for IgG, HSA, hemoglobin and HbA1c with VPBA-coated and uncoated SPR sensors.
Langmuir -Freundlich 12.787 601.023 0.00166 0.9288
ΔRmax 1/n KD,(mL/μg) KA,(μg/mL) R2
Measurements are made with artificial plasma to determine the selectivity of the boronic acid-based surface plasmon resonance sensor. Measurements are made to see whether the high affinity based on the cis-diol interaction of HbA1c to boronic acid shows equally high affinity for the IgG, HSA and Hb in plasma. The sensogram of all the different samples at a concentration of 120, 150, 200 μg/mL and the sensograms obtained by adding HbA1c at a concentration of 30, 50, 120 μg/mL to the artificial plasma are obtained and shown in Fig. 4. The comparison experiment of HbA1c binding signal with IgG, Hb, and HSA are
0.1105 9.0506 18.1811 0.05500 0.9244
6
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Fig. 5. Reusability sensogram of VPBA coated SPR sensor in HbA1c measurement.
conducted at the different concentration. According to the results, there are neglectable interaction between boronic acid and compare molecules. Selectivity coefficient (k) and relative selectivity coefficient (k´) of the prepared sensor are determined by subtracting from the 6th and 7th equations and shown in Table 6. k=ΔRHbA1c/ΔRcompetitor
(11)
k´=kVPBA/kempty
(12)
For HbA1c, the VPBA spiked sensor showed 7.32 times more selectivity than IgG and also showed 17.33 and 34.66 more selectivity according to HSA and Hb respectively. Additionally, when the ΔR comparison with the unmodified chip is examined, a serious difference of 5.2/0.15 is observed, which proves that nanofilm is successfully established high affinity with HbA1c. 3.4. Reusability studies In the reusability study, HbA1c solution in pH 6.0 buffer at a concentration of 75 μg/mL is given to the system at room temperature. After passing through the buffer system, the sample is given for about 5 min and after 3 min the desorption solution is passed, the same procedure is repeated 5 times and shown in Fig. 5. As shown in the figure, almost the same ΔR value is obtained for each repeated sample. 4. Conclusion Studies have been conducted to determine the glycosylated hemoglobin (HbA1c), which is a vital measure for diabetes detection and early diagnosis, with the SPR sensor. Because the cis-diol interactions between glucose and boronic acid are predicted to be unique and potent, vinyl phenyl boronic acid (VPBA) bound coated nanofilm is synthesized and the relationship between glycosylated hemoglobin is observed by experiments. All characterization studies and subsequent kinetic studies, equilibrium analysis and isotherms proves that the desired binding is occurred. The detection limit for the study is calculated as 2.86 μg/mL. Although this value is higher than the calculated limit values in other studies, it can be lowered by the concentration and device changes in the calibration stage. Finally, the proposed approach can perform measurements far below the aforementioned clinical values proving the validity of the study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 7
Sensors & Actuators: B. Chemical 306 (2020) 127561
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sensing of total and glycated haemoglobin: the potential of using a specific singlefrequency value for analysis, Anal. Chim. Acta 936 (2016) 1–11. [32] D.M. Kim, Y.B. Shim, Disposable amperometric glycated hemoglobin sensor for the finger prick blood test, Anal. Chem. 85 (2013) 6536–6543. [33] Y. Zhou, H. Dong, L. Liu, Y. Hao, Z. Chang, M. Xu, Fabrication of electrochemical interface based on boronic acid-modified pyrroloquinoline quinine/reduced graphene oxide composites for voltammetric determination of glycated hemoglobin, Biosens. Bioelectron. 64 (2015) 442–448.
purification and isolation of biomolecules.
Merve Çalışır received master degree from Hacettepe University, Turkey in 2019. She is working on optical sensor system, protein detection, chromatographic separation of biomolecules and biosensors.
Adil Denizli (PhD) is a professor at Hacettepe University, Ankara, Turkey. He received PhD degree from the same university in 1992. He is a professor at the chemistry department, Hacettepe University since 2000 and now he is also the head of the Biochemistry Division. His main research fields are molecular imprinting technologies, hemoperfusion, removal of toxic materials from blood, purification of enzymes and proteins by chromatographic methods, biosensors based (SPR, QCM) on synthetic receptor technology, production of polymers havedifferent surface and bulk properties, shape and geometries, application of these polymers in medicine and biology.
Handan Yavuz (PhD) is a professor at Hacettepe University, Ankara, Turkey. She received PhD degree from the same university in 2003. She is a professor at Chemistry department, Hacettepe University. Her main research fields are preparation of polymeric biospecific sorbents and their use for the purification of enzymes and proteins, production of polymeric biomaterials, characterization of bulk and surface properties and modification of their surfaces by various methods.
Monireh Bakhshpour (PhD) received the master degree and PhD degree in 2011 and 2015 at Biochemistry Division of Chemistry Department, Hacettepe University respectively. She is working on designed and developed Surface Plasmon Resonance (SPR), and Quartz Crystal Microbalance (QCM) based chips for detection of pathogenic microorganisms, affinity chromatography, synthesis and characterization of polymeric matrix,
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